3,6,-Di-O-methyl-D-glucose has recently been recognized, isolated, and purified from specimens of Mycobacterium leprae, the vector for human leprosy [Brennan, et al., Int. J. Lepr. 48: 382-387 (1980); Hunter, et al., J. Bacteriol 147: 728-735 (1981); Hunter, et al., J. Biol. Chem. 257: 15072-15078 (1982); Tarelli, et al., Carbohydr. Res. 131: 346-352 (1984)]. This methylated sugar is a part of a phenolic glycolipid, known as PGL-I to those in the art. The lipid molecule contains a diacylated phenol phthiocerol, which is the definitive structural feature of certain "mycosides." "Mycosides," as used herein, refers to a group of glycoside containing molecules characteristic of several mycobacterial species [Asselineau, et al., Annales de Microbiologie 129: 46-69 (1978)]. PGL-I iss, however, distinct from all other similar mycosides in that its phenolic hydroxyl is glycosidically linked to a unique trisaccharide. This trisaccharide confers highly specific antigenic and serological activity to the lipid "backbone," which is generally inert.
The art recognizes that glycolipids may be used as serological diagnostic test reagents. Many tumor specific antigens are glycolipids. Further, and of specific interest to the invention described infra, is the efficacy of PGL-I as a reagent useful in recognizing leprosy infection or disease [Payne, et al., Int. J. Lepr. 50: 220-221]. The more specific phenolic glycolipid-I can be employed, e.g., in connection with enzyme linked immunosorbent assays (ELISAs), immunoenzymometric assays (IEMAs), radioimmunoassays (RIAs) or any of the diagnostic methods currently in use which involve detection or estimation of antibodies. PGL-I has been recognized as useful for detecting humoral antibodies specific for the contained haptenic di- or tri-saccharide moieties, Gigg, et al., Chem. Phys. Lipids 38: 299-307 (1985); Fujiwara, et al., Infect. Immun. 43: 245-252 (1984). Glycoconjugates of these with bovine serum albumin (BSA) have been prepared and used for this purpose (Fujiwara, supra). The rarity of PGL-I as a naturally occurring compound, as well as the obvious difficulty and expense of obtaining it in sufficient quantities for diagnostic assays, have, therefore, served as a stimulus for deriving methods of synthesizing the compound or of satisfactory alternatives. These include derivatives of the entire trisaccharide, the terminal disaccharide and even of the terminal 3,6-di-O-methyl glucose which is the principal specific antigen of the trisaccharide moiety (Fujiwara et al., supra).
Several synthesis methods for this methylated glucose are known to the art. Bell, et al., J. Chem. Soc. (1936): 1553-1554, pioneered in this regard by treating 1,2,0-isopropylidene-3-0-methyl-6-0-p-tolylsulfonyl-.alpha.-D-glucofuranos e with sodium methoxide, as well as by methylation of 1,2,0-isopropylidene-.alpha.-D-glucofuranose-5-nitrate. Both syntheses gave intermediates that were readily converted into the 3,6-di-O-methylglucose. Gigg, et al., J. Chem. Soc. pp. 82-86 (1966), methylated 5-0-benzyl-1,2,-0-isopropylidene-D-glucofuranose to provide another such intermediate. Most recently, Gigg, et al., Chem. Phys. Lipids 38: 299-307 (1985), synthesized the compound from 5-0-allyl-1,2-0-isopropylidene 3-0-methyl-.alpha.-D-glucofuranose.
Each of these synthesis methods is complex, expensive, and involves numerous steps. The method described herein presents a simpler, more practical method, using readily available D-glucurono-6,3-lactone as the starting substance.